Non-monotonic dose effects of in utero exposure to di(2-ethylhexyl) phthalate (DEHP) on testicular and serum testosterone and anogenital distance in male mouse fetuses

Abstract

Di(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental contaminant. Epidemiological studies suggest that DEHP decreases masculinization of male fetuses. Numerous rat studies report DEHP reduces fetal testosterone production at doses greatly exceeding human exposure. We fed pregnant CD-1 mice 0.5-500,000 μg/kg/day DEHP from gestation day (GD) 9-18 and examined mothers and male fetuses on GD 18. We assessed non-monotonic dose-response by adding a quadratic term to a simple linear regression model. Except at the 500,000 μg/kg/day dose, DEHP stimulated an increase in maternal and fetal serum testosterone and increased anogenital distance (AGD). Non-monotonic dose-response curves were noted for AGD and maternal, and testis testosterone (P values 0.013-0.021). Because data from our highest dose (500,000 μg/kg/day) did not differ significantly from controls, this dose could have been incorrectly assumed to be the NOAEL had we only tested very high doses, as is typical in studies for regulatory agencies.

Figure 1

Serum MEHP concentrations as a function of maternal oral administration of doses of DEHP in A) the low dose range (0, 0.5, 1, 5 and 500 μg/kg/day) or B) the high dose range (50 or 500 mg/kg/day). Fetal male serum concentrations are given for both low- and high-dose ranges and maternal serum MEHP concentrations are given for the high dose range only. Low-dose measurements were of single pooled serum samples from each treatment, and values represent the mean (±SEM) of duplicate measurements; in B) values represent the mean (±SEM) concentration of 5-6 individual serum samples at each data point.

Figure 2

Effect of different doses of DEHP on maternal serum testosterone concentrations on GD 18 (ANOVA on log-transformed data, P<0.05). Values represent the mean ±SEM. Treatments with the same letter are not significantly different from each other but are statistically different from groups with other letters. b*, P=0.07 relative to controls; b**, P = 0.09 relative to 500,000 group. Sample sizes were: Oil n=20, 0.5 μg n=9, 1 μg n=11, 5 μg n=12, 500 μg n=13, 50 mg n=16, 500 mg n=17.

Figure 3

Effect of different doses of DEHP on serum testosterone concentrations in fetal males on GD 18 (ANOVA on log-transformed data, P <0.05). Values represent the mean ±SEM. Treatments with the same letter are not significantly different from each other but are statistically different from groups with other letters. a vs. b, P<0.1; b vs. c, P<0.05, b*vs. c, P=0.08. Sample sizes were: Oil n=21, 0.5 μg n=12, 1 μg n=11, 5 μg n=11, 500 μg n=13, 50 mg n=16, 500 mg n=18.

Figure 4

Effect of DEHP on the ratio of 1MF male anogenital distance (AGD) to body weight (BW). Values represent the mean ±SEM. There was a significant effect of dose based on regression analysis that included a quadratic term (P < 0.05). Treatments with the same letter are not significantly different from each other but are statistically different from groups with other letters. Sample sizes were: Oil n=13, 0.5 μg n=5, 1 μg n=7, 5 μg n=9, 500 μg n=9, 50 mg n=13, 500 mg n=13.